Bacterial Profile of External Ocular Infections, Its Associated Factors, and Antimicrobial Susceptibility Pattern among Patients Attending Karamara Hospital, Jigjiga, Eastern Ethiopia

Background External ocular infection is a global public health problem. Frequently, bacteria cause an ocular infection that ranges from morbidity to loss of vision. The increasing bacterial resistance in ocular infections leads to the risk of treatment failure with possibly serious consequences. Objective The study aimed to assess the bacterial profile of external ocular infections, their associated factors, and antimicrobial susceptibility pattern among patients admitted to Karamara hospital, Jigjiga, Eastern Ethiopia. Method Institutional-basedcross-sectional study was conducted on 288 conveniently selected patients among patients admitted to Karamara hospital from May 1 to June 30, 2020. Data were collected using a structured questionnaire. The ocular sample was collected and cultured in the appropriate culture media and identified using a series of biochemical tests. Antimicrobial susceptibility testing of isolates was performed by using the disk diffusion method. Data were double entered onto EpiData version 3.1 then exported to SPSS version 20 and analyzed to calculate descriptive frequency and odds ratio, and p value ≤0.05 was taken as the significant value. Result The prevalence of bacterial infection in external ocular samples was 62.2% (95% CI: 56.6%, 68.4%). Out of the 179 isolates, the majority of the bacterial isolates (87.7%) were Gram-positive. Staphylococcus aureus (53.1%) was the predominant isolate. Using soap for washing the face (AOR = 0.43; 95% CI: 0.29, 0.95), having diabetes mellitus (AOR = 3.11; 95% CI: 1.45, 6.75), and history of hospitalization (AOR = 2.82; 95% CI: 1.44, 5.54) were significantly associated with external ocular infection. Most (95.5%) of the Gram-positive bacteria showed resistance to penicillin, but they were susceptible to vancomycin, clindamycin, and ciprofloxacin. Conclusion The study showed a high prevalence of bacterial infections with the predominant isolate was S. aureus. Penicillin-resistant bacteria were identified among Gram-positive bacterial isolates. Soap usage, hospitalization, and diabetes mellitus were associated with the infection. Antibiotics that were susceptible to the specific bacteria should be used as a drug of choice and using soap for washing the face is advisable to protect against external ocular infection.


Introduction
Microorganisms are closely associated with external ocular infection. Particularly, infections caused by bacteria are quite common [1]. Te most common external ocular infections include conjunctivitis, blepharitis, dacryocystitis, orbital, and periorbital cellulitis. Tese infections are among the leading causes of ocular morbidity and blindness worldwide, chiefy in developing countries like Ethiopia [2,3]. Despite considerable resident microbiota, the eye is exposed to an external environment where a range of microorganisms is also inhibited which can cause eye infections opportunistically [4]. Several bacteria play a great role in triggering eye infections and corneal [5,6]. Te common bacterial agents responsible for ocular infections include Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, and several Streptococcus and Bacillus spp. as well as Gram-negative bacteria such as Pseudomonas aeruginosa,

Study Area and Period.
Te study was conducted in Karamara hospital in Jigjiga from May 1 to June 30, 2020. Jigjiga town is found in the eastern part of Ethiopia, and it is the capital city of the Somali region. It is found 635 km away from Addis Ababa. Karamara hospital renders health services for over seven million people living in all zones and districts of the Somali region. It has high patient fow in the eye clinic.

Study Design and
Population. An institutionalbasedcross-sectional study was employed. Two hundred eighty-eight (288) patients who visited the eye clinic of Karamara hospital with suspected external ocular infections and fulflled the inclusion criteria during the study period were included. Patients on antibiotics, anti-infammatory drugs, and those diagnosed with allergic problems and trachoma were excluded.

Sample Size Determination and Sampling Techniques.
Te sample size of the study was determined using a single population proportion formula by considering the prevalence of bacterial pathogens among patients with external ocular infection (21%) from the study conducted in Hawassa University Teaching and Referral Hospital, southern Ethiopia [15], with 95% confdence interval (CI), 5% margin of error and 10% nonresponse rate. Ten, the fnal sample size was 288. Te study participants were recruited conveniently until we got the required sample size.

Physical Examination, Specimen Collection, and
Transportation. All patients suspected with external ocular infections were physically examined using a slit-lamp biomicroscope and diagnosed by an ophthalmologist. Specimens from the external part of the eye, such as conjunctiva, eyelid, and lacrimal sac, were taken by an ophthalmologist. Conjunctival specimens were collected using a sterile saline moistened cotton swab, applied by passing the swab gently over the lower and upper conjunctiva 2-times [21]. In cases of dacryocystitis, specimens were taken by puncture and aspiration of the lacrimal sac. An antiseptic was frst applied to the area of the puncture, and then the lacrimal sac was punctured in the area below the medial canthal ligament [22]. In the case of blepharitis, discharge from the margin of the eyelid was collected using cotton swabs and placed into a sterile tube. All the swabs were fnally immersed in a tube that had 3 ml brain heart infusion (BHI) [23] and transported to the Somali regional microbiology laboratory by using the cold box. After specimen collection, data on sociodemographic and associated factors with external ocular infection were collected by a trained optometrist from each study participant using a pretested structured questionnaire adapted from the previous studies [15,24].

Bacterial Isolation and Identifcation.
Gram staining was done for diferentiating Gram-positive and Gram-negative bacteria and to observe the presence and morphology of cells. Smears were prepared at the collection sites from swabs by gently circularly spreading the specimen on a glass slide [25]. Each specimen was inoculated on a blood agar plate (BAP), chocolate agar plate (CAP), MacConkey agar (MAC), and mannitol salt ager (MSA) culture media with sterile wire loops and incubated at 37°C for 48 hours. Chocolate agar plates were incubated within a candle-jar to facilitate the CO 2 atmosphere. After 24 hours of incubation, the plates were observed and examined for bacterial pathogen growth, and plates with no growth were reincubated for further 24 hours [26].
Te identifcation of bacterial pathogens was done initially by Gram stain and colony morphology from culture followed by biochemical tests. Biochemical tests like catalase, coagulase, optochin disk, and bile solubility tests were applied to identify and diferentiate Gram-positive cocci, while biochemical tests, such as triple sugar iron agar (TSI), citrate utilization, lysine decarboxylase agar (LDC), oxidase, urease, indole, Methyl Red-Voges-Proskauer (MR-VP), and tributyrin tests were used to identify Gram-negative bacterial isolates [26,27]. Grampositive bacteria were identifed using hemolytic activity on sheep blood agar, catalase for diferentiation of Gram-positive and Gram-negative, coagulase test for S. aureus, bile solubility, and optochin disk test sensitivity for S. pneumonia [26].

Antimicrobial Susceptibility
Testing. An antimicrobial susceptibility testing was carried out on each identifed bacterium using the disc difusion method on Mueller Hinton agar (MHA). Besides, MHA medium containing 5% defbrinated sheep blood was used for fastidious bacterial isolate like S. pneumoniae. Primarily, 3-5 bacterial colonies of the test organism were picked and emulsifed in 5 ml of nutrient broth and mixed gently. To standardize the density of the inoculum for a susceptibility test, a 0.5 McFarland standard solution was used. Te plates were then inoculated by streaking the swab over the entire agar surface then the common antimicrobials were used for patients treated in the Karamara general hospital with the following concentrations: ceftriaxone (CRO) (30 μg), ciprofoxacin (CIP) (5 μg), trimethoprim-sulfamethoxazole (SXT) (25 μg), gentamicin (CN) (10 μg), tetracycline (TE) (30 μg), penicillin (P) (10U) and clindamycin (DA) (2 μg), vancomycin (VA) (30 μg), and doxycycline (DO) (30 μg) were placed using sterile forceps on the plate's surface on the MHA plate and incubated at 37°C for 18-24 hours but for S. aureus, it was incubated for only 16-18 hours, and then the zone of inhibition was determined. Te zone diameters were measured and recorded. Finally, bacterial susceptibilities were interpreted following the Clinical and Laboratory Standards Institute (CLSI) guidelines as susceptible (S), intermediate (I), or resistant [28].

Quality
Control. Te reliability of this study was ensured by actualizing quality control (QC) measures all through the entire process of the laboratory procedures. All necessary materials, equipment, and procedures were controlled enough. Te questionnaire was prepared in English language and translated to Amharic and the Somali language then retranslated to English to check the consistency. Te data were collected by a trained optometrist. Needed specimens were collected following the standard operating procedures (SOPs) that were prepared specifcally for external ocular specimen collection. Culture media sterility was ensured by incubating uninoculated media. Te prepared culture media performance was checked by inoculating the standard strains, such as Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATCC 27853) [25,27] obtained from the Ethiopian Public Health Institute, Addis Ababa, Ethiopia. Tese strains were also used to check the qualities of biochemical tests. Furthermore, the quality of the data entry was maintained by double data entry.

Data
Analysis. Te data were cleaned, coded, and double entered using EpiData version 3.1 software and then exported to statistical package for Social Sciences version 20 software for analysis. Te descriptive statistics (mean, percentages or frequency) were calculated to summarize the fndings. Te magnitude of the association between the diferent variables to the outcome variables was measured by the odds ratio with a 95% confdence interval (CI). Bivariate and multivariable logistic regression analyses were performed to assess the association between dependent and independent variables. Crude odds ratio (COR) and adjusted odds ratio (AOR) at 95% confdence interval was used to measure the strength of association. Tose variables with p value <0.2 at bivariate logistic regression were considered for the multivariable logistic regression model to control the confding variables. Statistical signifcance was declared at a p value less than 0.05.

Ethical Consideration. Ethical clearance was obtained from the Institutional Health Research Ethics Review Committee (IHRERC) Health and Medical Sciences College, Haramaya
University. An ofcial permission letter was written to the Somali regional health ofce which wrote a permission letter to Karamara hospital. Te objective, purpose, risk, and benefts were explained and the signed consent was obtained from the hospital head, the study participants and guardian, or parents of children under 18 years. All the information obtained from the study participants were kept confdential but positive culture result with the possible drug of choice was reported to the ophthalmologist for proper treatment.

Sociodemographic Characteristics.
A total of 288 patients were clinically diagnosed with external ocular infections and included in this study with a response rate of 100%. About 52.8% of the study participants were males. Te mean age was 38.5 (SD ± 16.2) years and 49% of the study participants were of the age between 18 and 39 years. Approximately 71.2% of the participants were from urban and 31.3% were businessmen. One-fourth of the study participants had formal education up to the primary school level (Table 1).

Antimicrobial Susceptibility Patterns of Gram-Negative
Bacterial Isolate. Over 68% and 63% of Gram-negative bacteria isolates were sensitive to gentamicin and ceftriaxone, respectively. However, 50% and 77.3% were resistant to tetracycline and trimethoprim-sulfamethoxazole, respectively (Table 6).

Multi-Drug Resistance.
In this study, the overall multidrug resistance (resistance to two or more antimicrobials) was 87.7%. Only 2.2% were sensitive to all tested antimicrobials (Table 7).
Te current study showed a higher prevalence of conjunctivitis (58%) and blepharitis as the next most dominant types of eye infection (33.5%). Tis is consistent with a study conducted in northwest Ethiopia [15]. Staphylococcus aureus was the most common isolate in conjunctivitis (55.8%), blepharitis (50%), and blepharoconjunctivitis (40%). A similar conclusion was reached by studies conducted in Ethiopia [6] and India [33]. On the other hand, S. aureus was isolated from blepharitis (47.6%) and conjunctivitis (26.6%) as reported from northern Ethiopia [9]. Tis dominance of S. aureus might be due to contamination of the eye from skin normal fora as a result of touching the eyes with contaminated hands [37].
In the present study, those who used soap were less likely to develop an external ocular infection. It increases personal hygiene, which prevents the growth of bacterial pathogens on the exterior part of the eye, and it is supported by a similar study conducted in France [38]. However, another study reported that there is no signifcant association between soap usage and external ocular infection [15]. Tis protective association might be due to the chemical characteristics of soap, which destroys the pathogen from the infection site.
History of hospitalization was signifcantly associated with external ocular infection. Tis is consistent with the study conducted in Portugal [39] and in the USA, Central California [40]. Te main reason for this signifcant association is due to the characteristics of the bacteria that cause external ocular infections. Tese bacteria cause nosocomial infection, that can be acquired during hospitalization [41].
Being diabetes mellitus was signifcantly associated with ocular infection, this result is supported by several studies in China [42], Denmark [43], England [44], and Iran [3]. Tis is due to individuals with diabetes mellitus having lower immunity, which may result in loss of control for systemic infections with subsequent spread to ocular tissues [43].
Te drug susceptibility patterns of Gram-positive cocci bacterial isolates showed that sensitivity to vancomycin (96.8%) followed by ciprofoxacin (92.4%). Tis fnding agrees with studies conducted in Ethiopia [6] and India [33]. However, most of the isolates were resistant to penicillin and a similar pattern of results was obtained in Jimma [6] and Gondar [24]. Tis reduction in the efectiveness of penicillin could be due to the frequent usage that results from its low price and accessibility without a prescription.
Most of the Gram-negative isolates were sensitive to gentamicin (68%) followed by ceftriaxone (63%), but they were resistant to trimethoprim-sulphamethoxazole (78%). Several reports also showed similar patterns of drug resistance among Gram-negative bacteria Dessie, [15] Gondar, [20], and India [2]. Moraxella species were 100% sensitive to ciprofoxacin, gentamicin, and tetracycline. Tis might be due to the few numbers of isolated Moraxella species. Teir sensitivity to ciprofoxacin is consistent with the studies conducted in Jimma [6] and Hawassa [45]. However, they showed 100% resistance to trimethoprimsulphamethoxazole. Tis might be due to a few isolates of the species.
In this study, most bacterial isolates were resistant to penicillin. Tis might be due to the usage of those broadspectrum antimicrobial agents without taking appropriate diagnosis. Tis result is supported by the study conducted in Jimma [6]. Te prevalence of multidrug resistance (MDR) to two or more bacterial isolates to the commonly prescribed antimicrobials was observed in 87.7% of the isolates. Tis is consistent with what has been found in previous studies conducted in Gondar, northwest Ethiopia [18]. However, a lower prevalence of multidrug resistance was previously reported in Hawassa, south Ethiopia [34]. Tis may be due to the diference in type and generation of antibiotics that we used for susceptibility testing.

Conclusion
In this study bacterial external ocular infections are highly prevalent. Conjunctivitis was the dominant external eye infection followed by blepharitis. Gram-positive bacteria constitute more than eighty-fve percent of isolates with S. aureus being the most predominant ones. Vancomycin and clindamycin were the drugs of choice for Gram-positive bacterial isolate and gentamicin and ceftriaxone were the drugs for a Gram-negative bacterial isolate. Te prevalence of MDR to the commonly prescribed antimicrobials was very high. In this study, soap usage, hospitalization, and diabetes mellitus were statistically signifcant. Terefore, the community should keep themselves from systemic diseases like DM and practice good personal hygiene to minimize the probability of getting external ocular infections. Antibiotics that have high sensitivity for each bacterial isolate should be used as a drug of choice for patients with external ocular infection. Using soap for washing the face is advisable to protect against external ocular infection. Additionally, Somali regional health ofces should give health education to the community to minimize the efect of possible risk factors.

Data Availability
All the generated data and the analysis developed in this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.